Cross-communication between different signalling systems allows the integration of the great diversity of stimuli that a cell receives under varying physiological situations. The transactivation of epidermal growth factor receptor (EGFR)-dependent signalling pathways upon stimulation of G-protein-coupled receptors (GPCRs), which are critical for the mitogenic activity of ligands such as lysophosphatidic acid, endothelin, thrombin, bombesin and carbachol, provides evidence for such an interconnected communication network. Here we show that EGFR transactivation upon GPCR stimulation involves proHB-EGF and a metalloproteinase activity that is rapidly induced upon GPCR-ligand interaction. We show that inhibition of proHB-EGF processing blocks GPCR-induced EGFR transactivation and downstream signals. The pathophysiological significance of this mechanism is demonstrated by inhibition of constitutive EGFR activity upon treatment of PC3 prostate carcinoma cells with the metalloproteinase inhibitor batimastat. Together, our results establish a new mechanistic concept for cross-communication among different signalling systems.

Transduction of a mitogenic signal from the cell membrane to the nucleus involves the adapter proteins SHC and Grb2, which mediate activation of the Ras/mitogen-activated protein (MAP) kinase pathway. In contrast to receptor tyrosine kinases (RTKs), the signalling steps leading to Ras/MAP kinase activation by G-protein-coupled receptors (GPCRs) are still poorly characterized but appear to include beta gamma subunits of heterotrimeric G-proteins and as-yet unidentified tyrosine kinases. We report here that the epidermal growth factor receptor (EGFR) and the neu oncoprotein become rapidly tyrosine-phosphorylated upon stimulation of Rat-1 cells with the GPCR agonists endothelin-1, lysophosphatic acid and thrombin, suggesting that there is an intracellular mechanism for transactivation. Specific inhibition of EGFR function by either the selective tyrphostin AG1478 or a dominant-negative EGFR mutant suppressed MAP kinase activation and strongly inhibited induction of fos gene expression and DNA synthesis. Our results demonstrate a role for RTKs as downstream mediators in GPCR mitogenic signalling and suggest a ligand-independent mechanism of RTK activation through intracellular signal crosstalk.

The epidermal growth factor receptor (EGFR) tyrosine kinase recently was identified as providing a link to mitogen-activated protein kinase (MAPK) in response to G protein-coupled receptor (GPCR) agonists in Rat-1 fibroblasts. This cross-talk pathway is also established in other cell types such as HaCaT keratinocytes, primary mouse astrocytes and COS-7 cells. Transient expression of either G q -or G i -coupled receptors in COS-7 cells allowed GPCR agonist-induced EGFR transactivation, and lysophosphatidic acid (LPA)-generated signals involved the docking protein Gab1.

Amplification and/or overexpression of HER2/neu and HER3 genes have been implicated in the development of cancer in humans. The fact that these receptor tyrosine kinases (RTKs) are frequently coexpressed in tumor‐derived cell lines and that heterodimers form high affinity binding sites for heregulin (HRG) suggests a novel mechanism for signal definition, diversification or amplification. In cells expressing HER2 and HER3, tyrosine phosphorylation of HER3 is markedly increased upon exposure to recombinant HRG. ATP binding site mutants of HER2 and HER3 demonstrate transphosphorylation of HER3 by HER2, but not vice versa. HRG‐induced transphosphorylation of HER3 results in a substrate phosphorylation pattern distinct from HER2 cells and enhances association of the receptor with SHC and phosphoinositol 3‐kinase in transfected 293 and mammary carcinoma‐derived MCF‐7 cells. The physiological relevance of HER2/HER3 heterodimerization is demonstrated by HRG‐dependent transformation of NIH 3T3 cells coexpressing the two receptors. These findings demonstrate the acquisition of expanded signaling capacities for HER2 by HRG‐induced heterodimerization with HER3 and provide a molecular basis for the involvement of receptor heteroactivation in the development of human malignancies.

The class II phosphoinositide 3-kinases (PI3K) PI3K-C2␣ and PI3K-C2␤ are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2␣ and PI3K-C2␤ represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2␣ and PI3K-C2␤ immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca 2؉ as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2␣ is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2␤ was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2␤ with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2␤ occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2␤ was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3 phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.The binding of polypeptide growth factors to their cell surface receptors triggers the recruitment of numerous molecules to form a localized signaling complex at the plasma membrane. Translocation to the activated receptor from intracellular compartments and conformational and posttranslational modifications all contribute to activate many of the recruited secondary messenger molecules and thus perpetuate the signaling cascade (57). The accumulation of 3Ј phosphoinositides has been observed in numerous cell types following their stimulation with polypeptide growth factors, cytokines, and chemotactic agents (19,25). In quiescent cultures, levels of phosphatidylinositol(3,4)-bisphosphate [PtdIns(3,4)P 2 ] and phosphatidylinositol(3,4,5)-triphosphate [PtdIns(3,4,5)P 3 ] are low but increase rapidly in response to cell stimulation (54). Conseque...